Lactobacillus acidophilus- An Overview

What is Lactobacillus acidophilus?

Lactobacillus is a taxonomically diverse group of Gram-positive, non-sporing rods that are defined by the formation of lactic acid as the sole end product of carbohydrate metabolism. Lactobacillus acidophilus is one of the most important species of Lactobacillus which has been extensively used to produce probiotics with numerous health benefits.

• L. acidophilus is a part of the normal microbiota of human beings, colonizing different parts of the body like the oral cavity, gastrointestinal tract, and female genitourinary tract.
• Besides, these also occur in plant materials like silage and in foodstuffs and agricultural products.
• The occurrence of L. acidophilus frequently occurs in fermented milk products like cheese and yogurt and in fermented beverages like wine and cider.
• The presence of L. acidophilus in food can be associated with the enhancement of flavors or can be due to spoilage.
• Most Lactobacillus species are considered non-pathogenic microbes and some are even consumed as probiotics to prevent certain infections.
• However, some instances of bacteremia and other infections caused by Lactobacillus and L. acidophilus, in particular, have been observed.
• One of the most prominent features of the species is its ability to grow at comparatively low pH conditions with optimal growth at a pH value of 5.5.
• The species name ‘acidophilus’ is taken from two words; ‘acidum’ meaning acid and ‘philus’ meaning loving, indicating the tendency of the bacteria to survive in low pH conditions. 
• The bacteria was first isolated from infant feces in 1900 by Moro which was then named Bacillus acidophilus. The name was later changed to Lactobacillus acidophilus by Hansen and Mocquot in 1970.
• Because of the large number of bacteria that occur in food, L. acidophilus has been considered a safe organism. But recently, some incidences of lactobacillemia have been observed.
• The infections by L. acidophilus are sporadic and account only for about 0.05 to 0.48% of all cases of infective endocarditis and bacteremia. 
• L. acidophilus has several industrial applications ranging from their use as probiotics to treat certain mild conditions to use in food and beverage industries.

Classification of Lactobacillus acidophilus

• Lactobacillus acidophilus belongs to the family Lactobacillaceae based on the phylogenetic analysis of their 16S rRNA sequences and consists of three distinct genera; Lactobacillus, Paralactobacillus, and Pediococcus.
• Currently, the genus consists of about 96 species and 16 subspecies which is growing at the rate of six new species per annum.
• The initial classification of Lactobacillus was based on phenotypic and metabolic characteristics, but the method of classification has since been changed to molecular characteristics like DNA-DNA hybridization, 16S rRNA sequences, and GC content.
• Lactobacillus species can be further divided into different groups based on carbohydrate metabolism; obligately homofermentative, heterofermentative, and facultatively heterofermentative.
• The widespread species of the genus Lactobacillus can be differentiated into 14 different subgroups, of which the L. acidophilus group is one of the most prominent ones.
• L. acidophilus belongs to the L. acidophilus group which represents one of the most well-defined and in-depth branching Lactobacillus phylogenetic subgroups.
• The members of this group are classified on the basis of their DNA-DNA homology, and the genomic GC content ranges from 32% to 50%.

The following is the taxonomical classification of L. acidophilus:

Habitat of Lactobacillus acidophilus

• The occurrence of L. acidophilus frequently occurs in habitats with high carbohydrate content and thus, can colonize several natural habitats like plants, mucosal surface of animals, and carbohydrate-rich food.
• In the human body, the number of L. acidophilus is high in areas like the mouth, gut, and vaginal cavity. 
• The bacteria in such areas are responsible for maintaining the pH level of the body parts, which then protects from pathogenic organisms that cannot survive such pH levels.
• L. acidophilus colonizes the intestines of many other mammals like pigs, cattle, mice, and rats.
• The colonization of the vaginal cavity by L. acidophilus has shown to decrease colonization by a common vaginal pathogen, Candida albicans. It has been assumed that this is due to the colonization of the membrane by L. acidophilus which doesn’t allow a surface for C. albicans to attach.
• Besides, the next typical habitat of L. acidophilus is food products, mostly fermented milk products and beverages.
• The presence of L. acidophilus in food is either beneficial as it brings out desirable flavors or is harmful as it might result in food spoilage.
• The occurrence of L. acidophilus in milk products is due to the availability of lactose as a source of carbohydrate for its growth.
• L. acidophilus also occurs in silage or hay, which is given as feed to different domestic animals. The bacteria are involved in the fermentation of sugar present in the grass to prepare the silage.
• In some cases, L. acidophilus has also been isolated from manure where the bacteria doubles its population every 20 minutes under appropriate substrate and temperature conditions.

Morphology of Lactobacillus acidophilus

• The cells of L. acidophilus are large non-sporing rods that are Gram-positive, but old culture (>48hr) might be Gram-negative.
• The length of the rods and the degree of curvature depends on the age of the culture and the composition of the medium and oxygen tension. The length of the bacteria ranges between 0.6-0.9 × 1.5-6 µm in dimensions.
• The cells divide along a single plane, and the tendency of chain formation varies among strains and is dependent on factors like growth phase and pH of the medium.
• The cells are rods with rounded ends that can occur either singly or in pair or short chains.
• Asymmetrical division of cells might result in wrinkled chains and in some rare cases, ring formation.
• The bacteria develop a peritrichous flagellum depending on the medium and the age of culture. It might be observed during isolation but is lost after transfer to an artificial medium.
• In L. acidophilus and other homofermentative lactobacilli, internal granulation can be observed during Gram staining and methylene blue staining.
• The cell wall of L. acidophilus is a typical gram-positive cell wall with peptidoglycan of the type Lys-D-Asp type.
• The cell membrane is a typical lipid bilayer with integrated protein units, and the fluidity of the membrane might change with the changes in the environment.
• A membrane-bound teichoic acid is present, but cell wall-bond teichoic acid might be absent in some strains.
• The cytoplasm contains typical bacterial ribosomes and nucleoids, along with large mesosomes.
• Mesosomes are often formed by invaginations of the cytoplasmic membrane and are filled with tubulin. 

Cultural Characteristics of Lactobacillus acidophilus

• As the nutrient requirement of Lactobacillus is complex, the media for the isolation of these bacteria are also complex.
• An example of a non-selective medium for Lactobacillus is the MRS medium at the pH of 6.2 to 6.4. MRS medium is a medium of choice for the luxuriant growth of Lactobacillus from clinical samples.
• Acetate medium (SL) is a selective medium for the selective isolation of Lactobacillus.
• The presence of Tween 80 enhances the growth of L. acidophilus with a high content of acetate at pH 5.4.
• The bacteria do not produce a characteristic odor on culture media, but numerous volatile compounds are produced when it is present in food that results in either food spoilage or desired pleasant aroma of fermented food.
• The optimum temperature for the growth of L. acidophilus is between 30°C and 42°C, and the optimum growth temperature is 35°C.
• L. acidophilus on artificial culture media can survive a pH of 5-7, and the optimum growth is observed at the pH value of 5.5.
• The luxuriant growth of L. acidophilus can be seen under high oxygen tension as most strains are aerobic but some strains, especially those isolated from food samples, are facultatively anaerobic and grow well at reduced oxygen tension and increased CO2.
• Nutritional requirements of L. acidophilus include compounds like calcium pantothenate, folic acid, niacin, and riboflavin.
• The growth of L. acidophilus on liquid media like MRS broth occurs throughout the liquid, but the cells settle soon as the growth ceases.

The following are some cultural characteristics of L. acidophilus on different culture media:

a. Nutrient Agar (NA)

• The colonies of L. acidophilus on NA plates appear small with an entire margin and are convex, smooth, and glistening.
• The size of the colonies remains between 2-5mm, and the colonies are opaque without any pigments.
• Some strains isolated from food samples might be mucoid colonies due to the production of slime.

b. MRS Agar

• On MRS agar, L. acidophilus produces slightly opalescent colonies that are light to medium in color.
• Most strains produce opaque colonies that are convex, glistening, and smooth with an entire margin. However, some strains can produce rough colonies.
• A slight proteolytic activity can be observed in the form of clearing of the media due to the production of cell wall-bound and cell wall-released proteases.

c. Blood Agar (BA)

• The colony morphology of L. acidophilus in blood agar is small to medium grey-colored colonies that exhibit very weak β-hemolysis.
• The hemolysis produced by L. acidophilus is termed bleaching as they produce changes in the agar that resemble β-hemolysis bit the stromata of the corpuscles or blood cells remain intact. 

Biochemical Characteristics of Lactobacillus acidophilus

The biochemical characteristics of L. acidophilus can be tabulated as follows:

Fermentation

Enzymatic Reactions

Role of Lactobacillus acidophilus as Biopreservation resource

• Lactobacillus acidophilus has different mechanisms that allow the organism to compete against other microorganisms, some of which might even be pathogenic strains.
• It exerts antimicrobial activity against food-borne microorganisms by producing various inhibitory components that include organic acids, hydrogen peroxides, ammonia, bacteriocins, and diacetyl.
• The antimicrobial activity of L. acidophilus against other microbes can also be due to the production of lactic acid and hydrogen peroxide.

a. Organic acids

• The end product of homofermentation of carbohydrates includes organic acids like lactic acids, acetic acids, and propionic acids that help reduce the pH of the medium and suppress the growth of certain pathogens and spoilage bacteria.
• Organic acids work as antimicrobials that interfere with the maintenance of cell membrane integrity, inhibition of active transport, and reduction of intercellular pH.
• These compounds have a wide range of antimicrobial activity, and they inhibit both Gram-positive and Gram-negative bacteria.

b. Diacetyl and Aldehydes

• Lactobacillus acidophilus also produced flavor compounds like diacetyl and acetaldehydes that have some antimicrobial activity.
• Diacetyl is produced during citrate metabolism that provides the aroma and flavor to butter and other fermented milk products.
• Gram-negative bacteria and some yeasts are sensitive to diacetyl and acetaldehyde than Gram-positive bacteria.

c. Hydrogen Peroxide

• Hydrogen peroxide is produced by L. acidophilus by different mechanisms in the presence of oxygen.
• The antimicrobial action of hydrogen peroxide is the result of oxidation of sulfhydryl groups resulting in denaturation of various enzymes and the peroxidation of membrane lipids that increase membrane permeability.
• The presence of hydrogen peroxide can inhibit the growth of psychrotrophic and pathogenic microorganisms even in refrigeration temperatures.

d. Bacteriocins

• Bacteriocins produced by L. acidophilus are proteinaceous substances with bacteriocidal activity against different microorganisms.
• Bacteriocins are physiologically important as they provide a competitive advantage to a strain in an ecological niche that is also occupied by other microbes.
• The bacteria, when ingested by humans, enables them to compete with pathogens that might be present in the gastrointestinal tract.
• The bacteriocins produced by L. acidophilus are divided into four classes on the basis of their structure and the mode of action; Class I, Class II, Class III, Class IV.
• The Class I bacteriocins are called lantibiotics which are produced by the bacteria in response to the attack or colonization by other microbes/
• Class II bacteriocins are small heat-stable proteins that are non-lanthionine-containing peptides.
• Class III bacteriocins include larger proteins that are heat labile, whereas Class IV toxins are complex substances with lipid and carbohydrate moieties. Some are hydrophobic heat-stable proteins.
• Some examples of bacteriocins produced by L. acidophilus include Lactobacin B, lactacin F, acidocin A and acidocin B.
• Lactocin B acts on other members of the Lactobacillus genus which include L. bulgaricus, L. helveticus, L. lactis, and L. leichmannii.
• Lactacin F is a protein with activity against Enterococcus faecalis and L. fermentum.
• Acidocin A and B are active against pathogens like Listeria monocytogenes, Clostridium sporogenes, and Brochthriz thermosphacta.

Role of Lactobacillus acidophilus in food and dairy industries

• Lactobacillus acidophilus has been used in the dairy industry for the manufacture of acidophilus milk, yogurt, miru-miru, and kefir.
• Acidophilus milk is different from non-fermented milk in that the acidophilus milk is suitable for individuals that lack lactase enzymes as the lactose in acidophilus milk is hydrolyzed by β-galactosidase of L. acidophilus.
• Certain strains of L. acidophilus are used as starter culture during milk fermentation where they acidify milk to generate flavor and texture by the action of proteolytic enzymes.
• The use of L. acidophilus as a starter culture helps in the production of lactic acid from lactose which in most cases, is responsible for the coagulation of milk by lowering the pH of the milk.
• Different flavors of fermented milk products are due to the production of volatile metabolites along with the incorporation of carbon dioxide during metabolism.
• In cheese, the flavor is determined during the ripening process which depends on both the starter and non-started Lactobacillus species.
• L. acidophilus helps to promote faster ripening of cheeses like cheddar to reduce the incidence of bitterness.
• In yogurt, the flavor is brought about by the synergistic action of Streptococcus thermophilus and Lactobacillus species.
• Some of the common flavor compounds produced by L. acidophilus during milk fermentation include organic acids like acetic acids, propanoic acid, and other compounds like diacetyl, acetaldehyde, and acetoin.
• In the case of yogurt, the texture is brought about by the production of exopolysaccharides that act as viscosifying agents.
• Coagulation further enhances the texture of yogurt, resulting from the neutralization of the negative charges on the milk, which is also induced by L. acidophilus.
• L. acidophilus is also present in powder form in milk powder which has longer longevity and biopreservation properties.
• Besides milk and milk products, L. acidophilus is also involved in the formation of other fermented foods like soymilk, soy-based yogurt, kombucha, fermented vegetable juice, kimchi, sausages, and salami.

Role of Lactobacillus acidophilus in human health

• Many species of Lactobacillus are accepted as human probiotics, and some strains of L. acidophilus with multiple health benefits have also been described.
• Some of the common health benefits associated with the consumption of L. acidophilus are reduction of gastrointestinal symptoms in lactose-intolerant individuals, relief from constipation, treatment of infantile diarrhea, and activity against Helicobacter pylori.
• Various studies have demonstrated the role of L. acidophilus in the prevention of gastric inflammation by H. pylori infections.
• L. acidophilus plays an essential role in people having lactose-intolerance issues as the bacteria degrades lactose by the action of β-galactosidase.

a. Negative impacts

• Even though the bacteria have multiple health benefits, there have been some instances of infections caused by L. acidophilus. These infections are rarely food-borne and might occur in immunocompromised individuals where the bacteria acts as an opportunistic pathogen.
• L. acidophilus-associated infections include lactobacillemia, which under severe conditions, leads to endocarditis and bacteremia.
• Even though the exact mechanism of infections by L. acidophilus are not yet known, there are some possible indicators of pathogenicity like enzymatic action of the bacteria, bacterial translocation, mucin degradation, and platelet aggregation.
• Colonization of intestinal tracts by L. acidophilus is one of the most important categories while selecting L. acidophilus strains as probiotics.
• The same mechanisms allow the bacteria to attaché and adhere to the host tissue surface in the cases of infections.
•  Besides, some of the strains of L. acidophilus have amino acid decarboxylase activity producing biogenic amines that might be harmful to the host. 
• Production of different proteases that support colonization and dissemination of bacteria through the host tissue surface are evidence of the link between L. acidophilus and endocarditis.
• However, the infections caused by L. acidophilus are found to very rare from epidemiological studies and also occur in few individuals with some pre-existing conditions.

b. Positive impacts

• The most important positive effect of L. acidophilus on human health is the use of the bacteria as a probiotic.

Probiotic Lactobacillus acidophilus

• Probiotics are living microorganisms that, when administered in the appropriate amount provide a health benefit to the host.
• The significance of healthy gut microorganisms in preventing gastrointestinal infections has long been studied, but the use of probiotics as preventive and therapeutic agents has increased only recently.
• The selection of L, acidophilus as a probiotic is based on certain criteria; ability of the organism to survive through the upper gastrointestinal tract and reach the site of action, tolerance to human gastric juice, and antagonistic acidity against intestinal pathogens.
• L. acidophilus has the ability to stabilize and modulate the intestinal microbiota and can establish strong adherence to epithelial cells.
• Some of the common L. acidophilus containing dairy products include pasteurized milk, ice cream, cheeses, and fermented milk. Yogurt is a classic probiotic fermented product that has been used for many years.
•  Cheese also acts as a suitable carrier for live probiotic bacteria as it has low oxygen levels and high lipid content.

Benefits of L. acidophilus probiotics

a. Digestive benefits

• As L. acidophilus helps in the metabolism and break down of lactose, it helps to relieve the effects of lactose intolerance.
• Lactose intolerance results in some symptoms like intestinal pain, diarrhea, gas, and bloating, which are all minimized by the use of L. acidophilus probiotics.
• It helps in the flourishment of essential gut microbiota by releasing essential growth factors which further helps in the proper digestion of food.
• The effect of L. acidophilus in irritable bowel syndrome has also been studied. The symptoms associated with the syndrome are diarrhea, constipation, and bloating, all of which are highly reduced by the intake of L. acidophilus probiotic
• Besides, different factors like bacteriocins and metabolic products, induce antagonistic activity against intestinal pathogens.

b. Immunity

• L. acidophilus probiotics are also associated with boosting the immune system as they help maintain the population of normal microbiota and produce various compounds with antagonistic activity.
• It also helps in reducing inflammation of various sites of the body, especially the digestive tract.
• The bacteria is known to prevent infections by Helicobacter pylori which is a common agent of infection of the digestive system.
• The preservative action of L. acidophilus has been discussed before where the production of various metabolic products helps in the prevention of colonization by pathogenic bacteria.

c. Other benefits

• Other benefits of L. acidophilus probiotics include the prevention and treatment of vaginal yeast infections.
• When L. acidophilus is used along with the necessary antibiotics, the infections can be lost within a few days as opposed to several weeks.
• The bacteria also help improve the microbiota of the digestive tract in non-breast-fed infants.
• There have studies on the anti-tumor activity of probiotics as they contain a high amount of high-density lipoproteins.
• Besides, such probiotics also have been known to reduce symptoms of atopic dermatitis.

Side effects of Lactobacillus acidophilus probiotics

• Even though the use of L. acidophilus probiotics is deemed safe, there are some minimal side effects that are associated with the intake of such probiotics
• One of the common side effects of L. acidophilus probiotics and other similar probiotics is gas, bloating, and other mild digestive complaints.
• Some instances of rashes and acne might also be seen depending on the immune condition of the individual as probiotics can induce inflammation.
• These side effects are minor side effects that should be short-lived and disappear within 12-14 days. If, however, severe effects or lasting effects are observed, the use of probiotics should be stopped, and immediate help should be taken.

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